Diabetes and the female reproductive system.

Diabetes and the Female R e p ro d u c t i v e S y s t e m Anindita Nandi,

MD,

Leonid Poretsky,

MD*

KEYWORDS Diabetes in pregnancy Insulin and the ovary PCOS KEY POINTS Insulin and IGF-1 pathways are important in the development and maintenance of the female reproductive system. Insulin resistance is linked to varying degrees of ovulatory dysfunction, hyperandrogenism, and infertility. Aggressive treatment of hyperglycemia in pregnancy is important in optimizing maternal and fetal outcomes.

INTRODUCTION

The importance of insulin action in maintaining female reproductive function was suggested as early as 1925, with observations of ovarian hypofunction in patients with insulin-dependent diabetes mellitus (type 1 diabetes mellitus). Joslin and colleagues1 reported that girls with type 1 diabetes mellitus failed to develop menarche.2 Within 2 months to 1 year of insulin administration, however, menarche was observed in some of the girls. States of insulin resistance or hyperinsulinemia have also been associated with female reproductive abnormalities. In 1765, a description of females with signs of androgen excess and obesity (valde obesa et virili) was put forth by Morgagni.3 In 1921, Archard and Thiers described the plight of diabete des femmes a barbe (diabetes of the bearded woman).4 Since then, identification of altered menstruation, decreased fertility, and hirsuitism in women with syndromes of severe insulin resistance, such as Rabson-Mendenhall syndrome, type A insulin resistance syndrome, or type B insulin resistance syndrome, have linked insulin resistance with hyperandrogenism and abnormalities of female reproduction.5,6 Recently, the more prevalent condition of polycystic ovarian syndrome (PCOS) has supported this correlation.7

Disclosure: The authors have no conflict of interest to disclose. Division of Endocrinology and Metabolism, Beth Israel Medical Center, Albert Einstein College of Medicine, 317 East 17th Street, 7th Floor, New York, NY 10003, USA * Corresponding author. E-mail address: [email protected] Endocrinol Metab Clin N Am 42 (2013) 915–946 http://dx.doi.org/10.1016/j.ecl.2013.07.007 0889-8529/13/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved.

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INSULIN ACTION IN THE FEMALE REPRODUCTIVE SYSTEM Insulin and the Ovary

The insulin receptor is a heterotetramer consisting of 2 a subunits and 2 b subunits.8 Insulin binds to the extracellular a subunit. Subsequently, the b subunit becomes phosphorylated on tyrosine residues and acquires kinase activity. This phosphorylation initiates activation of a series of intracellular proteins, including insulin receptor substrate (IRS) proteins, phosphatidylinositide-3 kinase (PI3K), and mitogenactivated protein kinase (MAPK).8,9 The activation of this signaling cascade acts as an effector for insulin’s many metabolic effects in protein synthesis, lipogenesis, and carbohydrate metabolism. Perhaps the most important effect is facilitation of glucose transport through the plasma membrane mediated by translocation of intravesicular glucose transporter proteins (GLUTs) from the cytoplasm to the cell membrane. This process is mediated by the PI3K pathway. MAPK activation is thought to mediate the growth-promoting effects of insulin.8,9 An alternative signaling pathway for insulin action has been described. Insulin binding to the a subunit, independent of b-subunit activation, leads to the generation of inositol-glycan second messengers at the cell membrane. The role of this pathway in traditional metabolic effects of insulin has not been identified. It has been suggested that this alternative pathway may participate in regulation of ovarian steroidogenesis mediated by insulin. Definitive evidence for this, however, is lacking (Fig. 1).9 The classical target organs for insulin action are the muscle, liver, and adipose tissue. Demonstration of insulin receptor expression, however, in ovaries from both humans and animals supports a role for insulin action in the ovary. Insulin receptors are widely distributed throughout all ovarian components, including the granulosa, thecal, and stromal compartments.10,11 Studies of normal ovarian tissue have shown

Fig. 1. Once insulin binds to its receptor, a signaling cascade is activated. The main pathway involves binding to the insulin receptor a-subunit and activation of the b-subunit tyrosine kinase activity. This, in turn, leads to IRS-1 and IRS-2 phosphorylation and PI3K activation. Subsequently, GLUT are translocated to cell membranes to allow influx of glucose. An alternate pathway leads to generation of inositol glycans after insulin binding to its own receptor. This second pathway may be important in mediating steroidogenic effects of insulin action.

Diabetes and the Female Reproductive System

that insulin binds to its receptor in stromal and follicular components of the ovary. Complete inhibition of insulin binding by specific anti-insulin receptor antibodies and lack of such an effect with antibodies to the type 1 insulinlike growth factor (IGF-1) receptor demonstrate that insulin binds to its ovarian receptors. Moreover, insulin receptors extracted from ovarian stromal tissues were shown to be autophosphorylated by incubation with insulin, similar to insulin receptor activation in classic target tissues.11 Insulin has also been shown to bind to stromal tissues in ovaries from women with PCOS. Complete inhibition by anti-insulin receptor antibodies demonstrates the specificity of this interaction. Insulin binding to ovarian cell membranes from the luteal phase is similar to that in the follicular phase.10 Although it was shown that insulin specifically binds to its own receptor in the ovary, the question arose whether ovarian insulin receptor function and regulation were similar to those in classical target organs. Hyperinsulinemia is known to downregulate insulin receptor expression in classical target tissues.12–14 In vitro, exposure of stromal ovarian tissue to high-dose insulin eliminates specific insulin binding, whereas termination of exposure leads to the recovery of this activity within 4 hours.12 In vivo, induction of hyperinsulinemia is accompanied by decreased insulin binding in the ovary, indicating the down-regulation of insulin receptors. Labeled IGF-1 binding was increased, suggesting up-regulation of IGF-1 receptors by hyperinsulinemia.15 Therefore, in settings of insulin resistance, alternate pathways may become important players in the ovary. In human studies, a correlation has been seen in insulin binding to ovarian stromal tissue and circulating leukocytes in postmenopausal women. This relationship was not consistent in premenopausal women.9,12 It is plausible that in premenopausal women, other factors, such as circulating gonadotropins, gonadal steroids, and autocrine regulators (such as IGFs), may influence insulin receptor expression. Insulin effects on ovarian steroidogenesis have been studied both in vitro and in vivo. In vitro, insulin increases production of androgens, estrogens, and progesterone by granulosa and thecal cells.1,16 Because, in some studies, hyperinsulinemia is required to stimulate steroidogenesis, it has been debated whether this action is modulated through IGF-1 receptors. The ability to block steroidogenesis from both granulosa cells and theca cells obtained from women with PCOS with anti-insulin receptor antibodies but not with antibodies against IGF-1 receptors, however, indicates that insulin mediates these actions through its own receptor.17,18 It is possible that insulin’s effect on steroidogenesis is mediated through stimulation of aromatase activity.19 Insulin in some studies has also been shown to activate 17a-hydroxylase and enhance forskolin stimulation of 3b-hydroxysteroid dehydrogenase.20,21 It has not been clearly demonstrated that insulin acutely stimulates steroidogenesis in vivo. In some studies of PCOS patients, insulin levels have been correlated with testosterone levels. Other studies, however, have not consistently supported this relationship.22–24 Similarly, discrepant results in effect on androgen production have been noted with insulin infusion studies that have maintained euglycemic hyperinsulinemia for several hours.25–28 Studies suggest that insulin acts as a co-gonadotropin at the level of the ovary. It enhances steroidogenic responses to gonadotropins both in vivo and in vitro.19,29–31 Insulin also enhances human chorionic gonadotropin (hCG)-induced ovarian growth and cyst formation in experimental animals.8 In vivo, the degree of hyperinsulinemia in women with PCOS has been correlated with ovarian volume.32 Moreover, with gonadotropin hyperstimulation, the increase in ovarian dimensions is greater in hyperinsulinemic PCOS patients than in normoinsulinemic PCOS women.32 Insulin has been shown to decrease hepatic sex hormone–binding globulin (SHBG) production. This suppressive action may be an important player in increasing free testosterone in patients with hyperinsulinemia. An increase in SHBG can be seen in

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women with PCOS who are treated with insulin sensitizers.33,34 Another protein that is under the regulatory control of insulin is IGF binding protein (IGFBP)-1. Insulin inhibits production of this protein by the liver, thereby resulting in reduced circulating levels of IGFBP-1. Insulin also inhibits IGFBP-1 production by ovarian granulosa cells through activation of the insulin receptor. Decreased IGFBP-1 may increase circulating free IGF-1 levels.9 IGF Proteins

Insulin and IGF-1 are closely related peptides. They share sequence homology and have nearly identical 3-D structures.35 The hormone receptors also have significant homology, featuring a heterotetrameric structure where 2 a subunits serve as the hormone binding site and 2 b subunits become autophosphorylated and acquire tyrosine kinase activity. Insulin binds to the IGF-1 receptor with lower affinity than to its intrinsic receptor. Although insulin shares significant homology with IGF-2, it does not bind the IGF-2 receptor. IGF-1 is not able to propagate phosphorylation of the insulin receptor, which maintains its specificity for insulin.36 Unlike insulin receptor, IGF and IGF receptor expression shows a cell-specific and stage-specific distribution in the human ovary. IGF peptides enter the ovarian follicular fluid (FF) both from the general circulation and intraovarian production. IGF-1 is largely released from the circulation, however, whereas IGF-2 is obtained mainly from local ovarian cells. IGF-2 levels in FF are 8 times higher than IGF-1 levels.9 IGF-1 mRNA is barely detected in the adult ovary and excluded from the granulosa cell lineage. IGF-2 mRNA and protein are noted in thecal cells and perifollicular vessels in all follicles. In small antral follicles, it is detected in both granulosa and thecal cells. IGF-2 mRNA is secreted by the granulosa cells of preovulatory follicles and of those extracted after ovarian hyperstimulation.37,38 IGF-1 receptor expression is predominantly in granulosa cells and oocytes in dominant antral follicles. IGF-2 receptors are also expressed in the dominant follicles, localized to both the granulosa and thecal layers.37 By reverse transcription–polymerase chain reaction analysis, both types of receptors are also noted in the stromal components of the ovary.38 Growth hormone receptor deficiency does not interfere with abililty to ovulate, maintain fertility, and respond to superovulation, suggesting that IGF-1 action is not necessary for the ovulatory process in women.39 The predominant ligand produced by the human ovary is IGF-2. Most of the studies of IGF effects on ovarian function and steroidogenesis, however, have been done with IGF-1. IGF-1 stimulates DNA synthesis and estrogen secretion by granulosa and luteal cells. Like insulin it inhibits IGFBP-1 production and acts synergistically with gonadotropins to augment estrogen and progesterone production.38,39 In more recent studies, IGF-2 stimulates basal progesterone and estrogen secretion as well as aromatization of androgens. Similar to insulin and IGF-1, IGF-2 has been shown to inhibit IGFBP-1 production. Both IGF-1 and IGF-2 act on human thecal cells in vitro to enhance androgen production.9,40 IGFBPs are a family of homologous proteins that regulate bioavailability of the IGFs and, therefore, play an important role in regulating their actions. There are 6 IGFBPs, IGFBP-1 through IGFBP-6. Although all of the IGFBPs have been shown to inhibit IGF action by limiting their bioavailability, IGFBP-1 and IGFBP-3 can also have a stimulatory effect by forming a pool of slow-release IGFs. A subset of the IGFBPs also has IGF-independent actions, such as inhibition of DNA synthesis and alteration of cellular motility.41 IGFBPs are expressed by granulosa and thecal cells and are present in FF in most species. In situ hybridization studies have shown distinct patterns of IGFBP mRNA


expression in the ovary.42 For example, IGFBP-1 is expressed only in granulosa cells of dominant follicles; IGFBP-2 is expressed in granulosa cells of small antral follicles; and IGFBP-3 is expressed in the theca layer of all follicles and the granulosa layer of dominant follicles. Furthermore, production of each IGFBP by granulosa cells is uniquely regulated. IGFBP-1 is inhibited by follicle-stimulating hormone (FSH), insulin, IGF-1, and IGF-2 and increased by luteinizing hormone (LH), epidermal growth factor, prostaglandins, and phorbol esters. IGFBP-2 production, however, is negatively regulated by LH.40,43–45 IGFBPs that are found in FF may be produced locally in the ovary or may have originated in other organs, such as the liver. There are differential patterns of IGFBP levels in FF in androgen-dominant and estrogen-dominant FF.46,47 FF obtained after hyperstimualtion with menopausal gonadotropins followed by hCG has demonstrated a distinct pattern of IGFBP production, suggesting that the hormonal milieu regulates the pattern of IGFBP expression.48 IGFBP proteases are a superfamily of proteins that regulate IGFBP levels. They include several classes of proteases, including metalloproteinases, kallikreins, and cathepsins. IFGBP proteases are specific for IGFBP substrates. IGFBP-3 is most susceptible to proteolysis whereas IGFBP-1 is most resistant.49 IGFBP proteases activate partial degradation of the IGFBPs, leading to affinity for IGF proteins. This, in turn, allows increased binding to and activation of the IGF receptors. The relationships between the various components of the insulin-related ovarian regulatory system is shown in Fig. 2. Reproductive Phenotypes with Altered Insulin Signaling

The importance of insulin action in female reproductive function is suggested by the preservation of this effect in various species. Mutations in the insulin and the related

Fig. 2. The interactions between components of the insulin-related ovarian regulatory system, which involves insulin, IGF-I, IGF-II, and their receptors and IGFBPs.

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IGF-1 pathways have repeatedly resulted in alterations of the female hypothalamicpituitary-gonadal axis. In Caenorhabditis elegans, mutations in the insulin/IGF-1 receptor homolog, DAF-2, induce developmental arrest in the dauer stage with reduced fertility.50 Mutations in the Drosophila IRS protein, CHICO, cause female sterility accompanied by reduced growth and increased lipid storage.51 In mammals, female IRS-2 null mice are sterile. They have small anovulatory ovaries. The ovaries are resistant to superovulation with gonadotropins, suggesting an intrinsic ovarian defect. In addition, there seem be central abnormalities, because the pituitary size is decreased and expression of LH is low in these animals.52 Female mice with neuron-specific insulin receptor knockout have impaired follicular maturation secondary to altered hypothalamic control of LH secretion.53 Reconstitution of the brain, liver, and pancreas insulin signaling in insulin receptor knockout mice rescues the mice from neonatal death, prevents diabetes, and restores reproductive function.54 Despite this evidence that insulin/IGF-1 is important in the proper development and functioning of the reproductive system, the exact mechanism by which these pathways regulate gonadal function remains elusive.

OVULATORY FUNCTION IN TYPE 1 DIABETES MELLITUS AND TYPE 2 DIABETES MELLITUS Type 2 Diabetes Mellitus

Given the role of insulin in ovarian physiology (discussed previously), it is not surprising that irregular menstrual cycles or anovulatory cycles have been associated with both type 1 diabetes mellitus and type 2 diabetes mellitus. In type 2 diabetes mellitus, it is difficult to dissect how much the confounding obesity or insulin resistance contributes to development of oligomenorrhea. Pima Indians have one of the highest rates of type 2 diabetes mellitus and obesity among ethnic populations. A cross-sectional study in nonpregnant women showed a significantly higher prevalence of type 2 diabetes mellitus in women with menstrual irregularities. When adjusted for body mass index (BMI), however, this difference was no longer significant. A higher rate of diabetes was most strongly correlated in the least obese quartile of women with anovulatory cycles. This relationship persisted with adjustment for age and BMI but was no longer significant when adjusted for waist-to-hip ratio.55 Thus, the presence of obesity or abnormal fat distribution makes it difficult to assess the true relationship between ovulation and type 2 diabetes mellitus. The importance of insulin resistance, independent of obesity and hyperglycemia, in ovulatory function has also been shown. Lean, euglycemic mice with varying degrees of hyperinsulinemia demonstrate abnormal estrous cycles and follicular development in comparison with wild-type counterparts.56 The prospective Nurses Health Study II showed an increased risk of developing type 2 diabetes mellitus in women with irregular menstrual cycles. The relative risk was greater in women with higher BMI but was also persistent in nonobese women.57 A large prospective study in the Netherlands similarly demonstrated increased prevalence of hyperglycemia in women with menstrual irregularities but no increase in risk of hypertension, hyperlipidemia, or inflammatory markers. This cohort was also shown to have 28% increased risk of developing coronary heart disease in comparison with women with regular cycles.58 Until recently, adolescents were not commonly diagnosed with type 2 diabetes mellitus and insulin resistance. Studies have shown, however, that those who have a history of premature puberty are at higher risk of ultimately developing hyperinsulinemia and type 2 diabetes mellitus. They are also at higher risk of developing truncal and general obesity.59–62 Additionally, the earliest clinical change in patients who develop


PCOS may be premature puberty. It is important, therefore, to serially assess the metabolic status of these patients.63 Type 1 Diabetes Mellitus

Menstrual dysfunction has been extensively studied in patients with type 1 diabetes mellitus. Historically, before the discovery of insulin (discussed previously), young girls with type 1 diabetes mellitus failed to develop menarche. Early studies after insulin became more widely available, continued to report delays in the age of onset of menstruation in comparison with nondiabetic girls. Bergqvist and colleagues64 in 1954 reported an average age of menarche of 13.9 years in nondiabetic patients versus 15.0 in diabetic girls. In 1982, Djursing and colleagues65 reported that menarche occurred 1 year later in diabetic girls. This difference, however, was not found statistically significant. Since the introduction of intensive diabetes treatment after the Diabetes Control and Complications Trial, such differences have been greatly reduced. Recent studies, however, indicate a persistent delay in age of menarche in adolescents with type 1 diabetes mellitus despite the advancements in diabetes treatment. A large crosssectional study showed a later age of menarche in girls with type 1 diabetes mellitus than in normoglycemic peers (12.92 0.09 vs 12.32 0.18; P 5 .0062). Although those with higher BMI had earlier onset of menarche, there was no correlation with hemoglobin A1C (HbA1c) level. Similarly, there was no association of delayed menarche with episodes of diabetic ketoacidosis or hypoglycemia in the 2 years preceding menarche.66 Similar delays in the age of menarche (12.6 1.5 years vs 12.25 1.4; P 5 .01) were noted in a case control study with type 1 diabetic adolescents. This delay was not influenced by BMI or HbA1c.67 Maintenance of a functional reproductive system is reflected in the presence of regular ovulatory cycles. Menstrual irregularities, however, are common in women with type 1 diabetes mellitus. Approximately, one-third of these women have irregular cycles during their reproductive years.66 A higher prevalence of oligomenorrhea is seen in women with poor metabolic control as indicated by HbA1c.68 Other studies in adolescents, however, have not shown an increased effect of type 1 diabetes mellitus on ovulatory function.69 Women with type 1 diabetes have also been shown to have an earlier age of menopause in comparison to nondiabetic sisters and controls. The presence of type 1 diabetes mellitus, menstrual irregularity before the age of 30, and unilateral oophorectomy seem to independently affect age of menopause.70 There are many theories as to the cause of ovulatory dysfunction in women with type 1 diabetes mellitus. The data (discussed previously) suggest that although hyperglycemia likely contributes to these manifestations, other factors may be involved in the pathophysiology. States of chronic disease and physiologic stress, such as malnutrition, are associated with delayed puberty and menarche. It is possible that the metabolic stress of the onset of type 1 diabetes mellitus affects age of menarche in the girls.66 Increased catecholamine and dopamine release during periods of hyperglycemia may also suppress LH levels. Additionally, in mice with streptozotocin-induced diabetes, there is a hypogonadotrophic supresssion of LH and FSH. The mechanism for this effect is believed to involve abnormal gonadotropin-releasing hormone (GnRH) secretion.71 A subset of women with type 1 diabetes mellitus also seems to develop a hyperandrogenic state with oligomenorrhea similar to PCOS, a disease typically associated with insulin resistance. Clinical hyperandrogenism is noted in as many as 30% to 40% women with type 1 diabetes mellitus. Moreover, ultrasonographic evidence of polycystic ovaries is seen in 40% to 50% of adult type 1 diabetic women but only in

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13% of age-matched nondiabetic controls. The number of women using intensive insulin treatment was higher in women with evidence of PCOS.72,73 It is hypothesized that this syndrome results from exogenous hyperinsulinemia due to intensive glucose control regimens. Under physiologic conditions, the insulin produced by the pancreas undergoes first-pass metabolism through the liver. This eliminates 50% to 70% of insulin secreted by the pancreas. Type 1 diabetic patients do not experience this first-pass metabolism and thus have higher systemic insulin levels for similar glucose control. This may explain the high prevalence of the syndrome in type 1 diabetic women.72,73 The PCOS in type 1 diabetic women, however, differs from that in nondiabetic patients. There is a less pronounced hirsuitism and, although total testosterone and androstenedione levels are increased, SHBG, gonadotropin, estradiol and dehydroepiandrosterone sulfate (DHEAS) levels remain within the normal range. Portal insulin levels are important in down-regulation of SHBG synthesis. Because exogenous insulin does not reach high levels in the portal circulation, SHBG levels are not suppressed and, thus, free testosterone levels are not elevated. These changes are not associated with degree of glucose control. Therefore, it is unlikely that hyperglycemia plays a causative role in the PCOS presentation. Moreover, during adolescence, there is an increased degree of weight gain, fat mass, and insulin resistance that may also play a role in development of hyperandrogemia and oligomenorrhea in this age group.71,73 There are to date no conclusive studies as to the best modality of treatment of the PCOS presentation in type 1 diabetic women. SYNDROMES OF EXTREME INSULIN RESISTANCE

As discussed previously, syndromes of insulin resistance spanning the gamut of mild to severe have been associated with both hyperandrogenism and female reproductive abnormalities and infertility. Some of the rare forms of extreme insulin resistance/ hyperinsulinemia are reviewed first. Severe insulin resistance is defined as fasting plasma insulin levels above 50 mU/mL to 70 mU/mL or peak (post–oral glucose tolerance test [OGTT]) insulin levels above 350 mU/mL.74 Some of the hyperinsulinemic disorders that report insulin levels in this range are due to genetic mutations in the insulin receptor gene, such as type A syndrome, leprechaunism, and Rabson-Mendenhall syndrome. In other syndromes, antibodies against the insulin receptor can be detected as in the type B syndrome.75 Despite the differing mechanisms of decreased insulin action, these syndromes of severe insulin resistance share similar clinical manifestations. There are both symptoms resulting from deficiency of insulin action at target organs and those resulting from high levels of circulating insulin. Insulin resistance at traditional target organs, such as the liver, and fat can lead to impaired glucose homeostasis and varying degrees of lipoatrophy. The diabetes in these patients can often be resistant to insulin action, requiring at times thousands of units of insulin daily to lower glucose levels. In syndromes that result from autoantibodies to the insulin receptor, however, hypoglycemia can result from activation of the insulin receptor by a stimulating antibody clone.74,75 The presence of acanthosis nigricans and ovarian hyperandrogenism has been attributed to the severe hyperinsulinemia. Acanthosis nigricans is a hyperpigmented lesion that usually occurs in the back of the neck and in the axilla. It is characterized by hyperkeratosis and epidermal papillomatosis. Acanthosis is present in all congenital syndromes of severe insulin resistance and is correlated with the degree of hyperinsulinemia. The exact mechanism leading to acanthosis nigricans has not been fully


delineated. It is attributed in part to insulin binding to IGF-1 receptors.75 Hyperandrogenism is a common feature in women with extreme insulin resistance syndromes. Studies suggest an ovarian source of hyperandrogenism rather than an adrenal source. Taylor and coworkers reported that only premenopausal women with type B syndrome develop hyperandrogenism.76,77 Subsequently, selective catheterization of the adrenal and ovarian veins showed significantly higher androgen levels in ovarian veins than in adrenal veins. Fasting insulin levels are correlated with ovarian volume. High levels of insulin have also been shown to stimulate androgen-producing cells in the ovary.75,78 Although a correlation between insulin resistance and hyperandrogenism has been established, it is debated which of these states develops first. Several studies support the hypothesis that hyperandrogenism leads to insulin resistance.75 Givens and associates79 reported that resection of a luteoma in a PCOS patient led to the regression of acanthosis nigricans, a marker of insulin resistance. Shoupe and Lobo80 showed that antagonism of androgens with spironolactone leads to decreased fasting insulin levels in patients with PCOS. Insulin resistance is a common occurrence during puberty, suggesting that sex hormones contribute to this state.81 Other studies, however, fail to support the role of hyperandrogenism in the development of insulin resistance. Administration of androgens does not affect insulin sensitivity in normal men.82 Billiar and colleagues83 demonstrated that sustained levels of hyperandrogenism in female monkeys did not alter insulin sensitivity. The effect of insulin resistance on development of hyperandrogenism is discussed later in the section on PCOS. Because of the presence of extreme insulin resistance, it is a challenge to successfully treat these patients. Diet and exercise have less efficacy in patients with extreme insulin resistance than in typical type 2 diabetes mellitus patients. Insulin sensitizers, including metformin and thiazolidinediones, have been shown to improve glycemia in type B syndrome and lipoatrophic diabetes.74,75 Insulin, despite use of high doses, often fails to provide adequate glycemic control. Similarly, sulfonylureas have shown limited benefit in this patient population.74 A few studies have looked at the effects of IGF-1 administration on glycemic control. Although short-term improvements in metabolic parameters were seen in several syndromes of severe insulin resistance, longerterm trials failed to show sustained improvements. Additionally, IGF-1 administration can be associated with side effects, such as fluid retention, carpal tunnel syndrome, and jaw pain. There is also some concern that IGF-1 may increase the risk of retinopathy and breast and prostate cancers.84 In patients with autoantibody-mediated insulin resistance, such as type B syndrome, immunomodulation has been explored as a modality of treatment. One suggested combination is short-term suppression with plasmapheresis and cyclophosphamide, with longer-term maintenance using cyclosporin A and azathioprine.74,75 POLYCYSTIC OVARY SYNDROME

Milder states of insulin resistance have also been associated with abnormalities of female fertility. The Stein-Leventhal syndrome was described as a combination of “enlarged sclerotic ovaries” associated with obesity, hirsuitism, irregular menstruation, and infertility.7,85 PCOS may be defined by 1 of 3 sets of criteria. Of the 3, the definition most commonly used was adapted by the National Institutes of Health (NIH), which defines PCOS by the presence of (1) hyperandrogenism, (2) ovulatory dysfunction, and (3) exclusion of disorders, such as hyperprolactinemia, thyroid disorders, and congenital adrenal hyperplasia. The Rotterdam criteria, alternatively, require the presence of 2 of the 3 following conditions: hyperandrogenism, ovulatory dysfunction, and

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polycystic ovaries on ultrasound. In 2005, the Androgen Excess and PCOS Society put forth a third set of consensus criteria that define PCOS as the presence of hyperandrogenism, along with either or both ovarian dysfunction and polycystic ovaries.86,87 Prevalence

The prevalence of PCOS ranges from 4% to 10% of the female population. The differences in criteria used to diagnose PCOS likely account for the variability in analyzing studies related to PCOS. Use of the Rotterdam criteria rather than the NIH criteria leads to the identification of 2 to 5 times larger cohort of women with this diagnosis.7 It is intriguing that a disease that actually decreases female fertility would have such a high prevalence rate within the general population. In genome-wide searches, many of the genes associated with or linked to PCOS fall within the category of metabolism and downstream targets of insulin action. Some of these genes lead to the thrifty phenotype, a group of cellular and physiologic changes that gear the body to conserve energy. This may have provided survival advantage during periods of famine, flooding, or other conditions that decrease food sources.88 The PCOS phenotype may also have conferred economic and health advantages by providing fewer family members to feed at a time of contraceptive unavailability and by spacing children to ease the burden on the mother’s health. Clinical Presentation

The clinical signs and symptoms of PCOS are varied; 60% of women have evidence of hirsuitism (increased hair growth), acne, and alopecia. Clinically, this syndrome is accompanied by oligomenorrhea or anovulatory cycles and signs of insulin resistance, such as acanthosis nigricans. These velvety hyperkeratotic plaques are directly correlated with the degree of insulin resistance.7 PCOS is strongly associated with metabolic and cardiovascular diseases outside of the reproductive axis. Obesity is strongly correlated with PCOS, with prevalence ranging between 30% and 75 % in some series.89,90 Women in the United States tend to have higher body weight than European counterparts. Visceral adiposity, as measured by increased waist circumference or waistto-hip ratio, has been linked to hyperandrogenism, insulin resistance, and glucose intolerance; 30% to 40 % of women with PCOS have impaired glucose tolerance and as many as 10% have type 2 diabetes mellitus by their fourth decade. Dunaif and colleagues91 demonstrated that women with PCOS have greater insulin resistance than normal women, even when matched for BMI and body fat distribution.89 Hypertension develops in some women with PCOS early during the reproductive years. This is often accompanied by reduced vascular compliance and endothelial dysfunction.92 Insulin-lowering therapy seems to improve endothelial function.93 Women with PCOS also have an increased prevalence of macrovascular disease and thrombosis. Lipid changes reflect increased triglycerides and decreased highdensity lipoprotein, the pattern most frequently seen in insulin resistance and type 2 diabetes mellitus.89 Women with PCOS additionally have an increased risk of endometrial hyperplasia and cancer. This is likely due to prolonged stimulation of endometrial tissue by estrogen without opposing progesterone effects. Breast and ovarian cancer have also been variably associated with PCOS.94 Biochemical and Laboratory Testing

Biochemically PCOS is associated with elevated androgens (total and free testosterone) in 80% to 90% of cases. DHEAS can also be elevated in 25% of women with polycystic ovaries. These biochemical changes are frequently associated with


decreased SHBG levels, resulting in elevated free testosterone with normal to mildly elevated total testosterone levels. Changes in steroid hormone levels are accompanied by alterations in gonadotropins. It is believed that increased responsiveness of gonadotropins to GnRH leads to increased amplitude and pulse frequency of LH secretion. This effect is independent of obesity in PCOS patients. Increased LH levels lead to ovarian thecal hypertrophy and subsequently increased androgen production. FSH levels, alternatively, are normal to low, resulting in an increased LH-to-FSH ratio. This phenomenon is due to negative feedback from estrogen, aromatized from increased amounts of testosterone.87,95,96 Pathophysiology

Insulin resistance is strongly correlated with PCOS. Euglycemic hyperinsulinemic glucose clamp studies have demonstrated significant reduction in insulin mediated glucose disposal in PCOS subjects, independent of obesity.87 Thus, lean PCOS patients have greater insulin resistance than lean normal counterparts. Increased body fat has a synergistic negative effect on insulin sensistivity.90 Insulin reistance may influence PCOS pathology through insulin’s direct actions on the ovary. As discussed previously, in vitro studies have shown stimulation of androgen production by incubation of ovarian thecal cells with insulin.16 Moreover, exposure of ovaries to insulin and hCG synergistically leads to ovarian growth and cyst formation (Fig. 3).87,97 Hyperinsulinemia and obesity are both also associated with a state of low-grade inflammation.98 Elevations are noted in cytokines (interleukin [IL]-6 and tumor necrosis factor [TNF]-a), chemokines (IL-18 and monocyte chemoattractant protein [MCP]-1) and adipokines (leptin and resistin). Systematic inflammation is believed to lead to recruitment of macrophages to the adipose tissue.99 Long-term consequences of the increased inflammatory state include increased risk of development of hypertension, dyslipidemia, and endothelial dysfunction. Endothelial dysfunction is partially due to inhibitory effects of cytokines on expression and activity of endothelial nitric oxide synthase, decreasing nitric oxide synthesis and vasodilation.98 Insulin resistance is noted in 50% to 70% of PCOS patients. It is not surprising, then, that positive correlations have also been noted between PCOS and elevated cytokines, such as C-reactive protein, IL-18, and TNF-a. Markers of lymphocytic and monocytic activation, such as IL-6, MCP-1, and migration inhibitory factor, have also been correlated with PCOS. Moreover, insulin sensitizer use has decreased these inflammatory markers.100 PCOS has also been associated with adhesion molecules and markers of endothelial dysfunction, such as soluble intercellular adhesion molecule 1 and soluble vascular adhesion molecule 1. These markers have been correlated with both the degree of insulin resistance and hyperandrogenemia.98

Fig. 3. Insulin acts as a co-gonadotropin at the level of the ovary. Greater ovarian growth and cyst formation is seen after 23 days of combined hCG, insulin, and GnRH antagonist (GnRHant) injections than insulin or hCG alone in Sprague-Dawley rats. Normal saline served as the control injection.

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In vitro data show that TNF-a stimulates proliferation and androgen production by thecal cells. TNF-a has also been shown to be synergistic with similar effects of insulin and IGF-1.101,102 Il-6, another cytokine, stimulates adrenal cells in vitro, increasing adrenal androgen steroidogenesis.103 Alternatively, androgens may stimulate low-grade inflammation through their effects on adipocyte biology. Androgens stimulate adipocyte hypertrophy and differentiation of preadipocytes into mature adipocytes.104 Androgens also increase lipolysis, leading to increased flux of free fatty acids into circulation.105 Insulin may directly affect the pituitary gland by increasing gonadotroph sensitivity to GnRH. This has been demonstrated in several in vitro studies with cultured pituitary cells.106,107 Moreover, treatment of PCOS with insulin sensitizers decreases LH levels. Insulin also alters several key regulators of signal transduction and function in the ovary. Studies in hyperinsulinemic experimental animals and women with PCOS show decreased insulin receptors, but increased IGF-1 receptors in the ovary.108,109 As discussed previously, insulin additionally inhibits IGFBP-1 production, leading to higher circulating and intraovarian levels of free IGF-1. Furthermore, insulin not only increases the expression of peroxisome-proliferator-activated receptor g (PPAR-g) in vitro in ovarian cells but also activates the steroidogenic acute regulatory protein (StAR) protein, through which PPAR-g enhances steroidogenesis (Fig. 4).87 Treatment of PCOS

PCOS is manifested by a conglomeration of symptoms that have been previously reviewed. These include hyperandrogenism, anovulation or oligomenorrhea, infertility,

Fig. 4. After interaction with its own receptor, insulin stimulates StAR protein expression via activation of insulin signaling cascade protein, IRS-1. Insulin can also amplify StAR protein expression by activation of PPAR-g expression. StAR protein expression leads to steroidogenesis in human ovarian cells.


and insulin resistance or elevations of glucose. The treatment armamentarium for PCOS addresses one or more of these manifestations of PCOS; they are reviewed sequentially. Because of the complexity of the topic of ovulation induction, however, it is reviewed separately in this article. One of the defining clinical signs of PCOS is hyperandrogenism. Hirsuitism can be addressed with depilatories, shaving, waxing, and electrolysis. Antiandrogenic agents can also be utilized. The most commonly used agent is spironolactone, an antimineralocorticoid with antiandrogenic activity. Others in this category include cyproterone acetate (a competitive inhibitor of testosterone and 5a-dihydrotestosterone binding to the androgen receptor), flutamide (a nonsteroidal antiandrogen), and finasteride (a 5a-reductase activity inhibitor). These agents can have feminizing effects on the male fetus and thus should be avoided in pregnancy.89,110 Oral contraceptives (OCPs) can also reduce hyperandrogenism by direct negative feedback on LH secretion, resulting in reduced androgen production by thecal cells. OCPs also stimulate the production of SHBG by the liver, leading to decreased free testosterone levels.89,110 In choosing an OCP, it is advisable to select a progestin with low androgenic potential, such as norethindrone, desogestrel, norgestimate, and drosperinone. There are conflicting data as to whether drospirenone is associated with increased risk of venous thromboembolism.111–114 Insulin sensitizers, such as metformin and thiazolidinediones, are effective agents in reducing circulating androgen levels. Metformin is a biguanide that functions by activating adenosine monophosphate–activated protein kinase. This results in decreased hepatic gluconeogenesis and increased insulin sensitivity.115,116 Metformin’s effect on hormonal changes in PCOS is mediated through its systemic effects on hyperinsulinemia and insulin sensitivity.87 Thiazolidinediones are PPAR-g agonists. They improve insulin sensitivity at the level of the skeletal muscle and adipocytes. Thiazolidinediones also have direct effects on ovarian steroid production, independent of improvement in insulin resistance.79 Several of these agents, in addition to lowering androgen levels, have greater metabolic and hormonal effects that decrease insulin resistance and improve ovulation rates in women. Prior to prescribing medications, however, a trial of lifestyle changes and weight management is an acceptable option. Reductions of 5% to 10% in weight can improve hyperinsulinemia, circulating androgens, ovulation, and pregnancy rate. Even more modest weight loss of 2% to 5% can restore ovulation. Very low calorie diets (350 kcal/d) for 4 weeks can reduce fasting insulin and free testosterone levels.117,118 Surgical weight loss with gastric banding, gastroplasty, or Roux-en-Y gastric bypass can also be considered. In a group of 12 patients with PCOS, bariatric surgery restored regular menstrual cycles in all patients. In a group of 17 patients with PCOS, weight loss surgery led to improvement in ovulation rates, insulin resistance, and hyperandrogenism in 12 months.110,119 It is postulated that the primary mechanism by which weight loss improves reproductive outcomes is by reduction of circulating insulin levels.118 Insulin sensitizers are not only important in improving hyperinsulinemia, insulin sensitivity, glucose dysregulation, and dyslipidemia but also in ameliorating the lowgrade inflammatory state seen in insulin resistance and PCOS.98,100 Changes in systemic metabolic parameters and direct insulin effects in the ovary lead to improved ovulation rates and normalization of hormonal changes with the use of metformin.120 A study comparing pioglitazone and metformin in obese PCOS patients showed similar improvements in insulin resistance.121 Thiazolidinediones have also been shown to improve dyslipidemia and have beneficial effects on proinflammatory and prothrombotic markers, such as TNF-a,

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plasminogen activator inhibitor type 1, and IL-6.122 Thiazolidinediones have both direct ovarian effects, mediated through the PPAR-g and the StAR protein, and indirect effects, mediated via insulin-independent action on the ovary (Table 1). Insulin-independent effects result in increased progesterone production and lower testosterone and estradiol levels. Decreased total and free testosterone with increased SHBG levels result from systemic changes in insulin levels induced by thiazolidinediones.87,123,124 In comparison with metformin, thiazolidinediones have similar effects on ovulation rates.121 There are caveats with the use of thiazolidinediones, however. Rosiglitazone has been reported to be associated with increased risk of myocardial infarction.125 There may be an increased risk of bladder cancer with the use of pioglitazone.126 Moreover, thiazolidinediones are linked to increased bone loss and risk of fractures in women with diabetes.127 Pioglitazone and rosiglitazone may affect bone metabolism by inhibiting osteoblast growth, increasing precursor differentiation into adipocytes, increasing fatty acid uptake, and reducing both alkaline phosphatase and osteocalcin activity.128 Glucagon-like peptide-1 (GLP-1) agonists are also being studied in PCOS patients. These agents enhance glucose-dependent insulin secretion, delay gastric emptying, and reduce appetite. A recent study compared exenatide versus metformin versus combination therapy in obese PCOS patients for 24 weeks. Higher ovulation rates were seen in the exenatide and combination groups: exenatide (50%), metformin (29%), and combination (86%). Greater weight loss was also noted in the exenatide and combination groups.129 When monotherapy is not effective, combination therapies can be considered. Comparison of metformin and OCP (ethinyl estradiol–cyproterone acetate) versus OCP showed greater decrease in androstenedione and increase in SHBG levels. There was, however, greater increase in cholesterol with the OCP alone group.86,130 Similarly, rosiglitazone and OCP showed greater decrease in androgen levels and increase in SHBG. Here, combination therapy resulted in higher decreased high-density lipoprotein levels than monotherapy.87,131 Ovulation Induction in PCOS

PCOS is a major cause of subfertility in the United States. To conceive, many women require ovulation induction or in vitro fertilization (IVF). The treatment options for induction of ovulation are reviewed. Clomiphene citrate is a synthetic triphenylethyne, a

Table 1 Effects of thiazolidinediones in ovarian function Direct: Can be Observed in Vitro; may be Present in Vivo

Indirect: Observed in Vivo; are due to Systemic Insulin-sensitizing Action and Reduction of Hyperinsulinemia

Insulin independent [ Progesterone

Y Testosterone

Y Testosterone

[ IGFBP-1

Y Estradiol

[ SHBG

[ IGFBP-1 (in the absence of insulin)

Y Free testosterone

Insulin sensitizing (enhanced insulin effect) Y IGFBP-1 production [ Estradiol production (in vivo, in a setting of high-dose insulin infusion)


partial estrogen receptor antagonist with antiestrogenic effects in the hypothalamus that interfere with the negative feedback from estrogen resulting in increased FSH levels, which in turn lead to follicular growth.87,132 Because of the high rate of successful ovulation and cost effectiveness of this treatment, it is considered first-line treatment of ovulation induction. The live birth rate after 6 months of clomiphene citrate treatment ranges from 20% to 40%. There is a discrepancy between ovulation rate (60%–85%) and pregnancy rate (30%–40%). This may be due to clomiphene’s antiestrogenic effects on the uterus. The risks associated with clomiphene citrate use include multiple pregnancies and ovarian hyperstimulation syndrome (OHSS).132 In 2007, a large prospective randomized controlled trial in 626 anovulatory, infertile women with PCOS showed higher rates of live birth in the clomiphene (22.5%) or combined metformin and clomiphene group (26.8%) compared with the metformin alone group (7.2%). Currently combination therapy is recommended in the subset of patients with BMI greater than 35, glucose intolerance, and clomiphene citrate resistance.133 Thus, although improved pregnancy rates are seen with metformin versus placebo, no improvement is seen with live birth rates. In clomiphene-resistant patients, a combination of metformin and clomiphene citrate significantly improves ovulation and pregnancy rates but, importantly, not the rate of live births. Metformin as a single agent does not show any benefit in clomiphene-resistant patients.110,134 Metformin, however, may improve oocyte and embryo quality in clomiphene citrate patients undergoing in IVF.135 Thiazolidinediones enhance spontaneous and clomiphene-induced ovulation. Fetal safety for these agents has not been established, however. Thus, they should be stopped in case of pregnancy.121 If patients are clomiphene-resistant, in addition to insulin sensitizers several other agents can be considered for fertility treatment. Administration of exogenous FSH can be effective but is an expensive treatment option that requires frequent monitoring with estradiol levels and ultrasound.110,118 The addition of clomiphene citrate to gonadotropins and GnRH antagonists leads to decreased rates of OHSS without evidence of effect on live birth and pregnancy.136 Aromatase inhibitors block peripheral conversion of androgens to estrogens. This reduction in estrogens increases FSH production and optimizes ovulation. In a prospective randomized trial, no difference in pregnancy rates between letrozole and clomiphene citrate was noted. Despite concern for teratogenicity, there was no increase in incidence of malformations.135 Another study comparing anastrazole and clomiphene citrate demonstrated thicker endometrium, fewer mature follicles, and a higher rate of pregnancy (nonsignificant) in the aromatase inhibitor arm.137 The efficacy and safety of these agents in comparison with clomiphene citrate in achieving live births may be more definitively answered in an ongoing trial, Pregnancy in Polycystic Ovary Syndrome II (PPCOS II).138 If treatment with clomiphene citrate, gonadotropins, and aromatase inhibitors is not successful, in vitro fertilization is recommended. There is an increased risk of multiple gestations with this treatment. The risk decreases, however, with single embryo transfer. A meta-analysis in 2006 showed that women with PCOS undergo greater numbers of cycle cancellations and increased duration of stimulation cycle compared with the general IVF population.139 Addition of clomiphene citrate may improve IVF outcomes.140 Coincident use of metformin increases viable pregnancy rates and decreases OHSS.118 Glucocorticoids, such as prednisone and dexamethasone, can suppress adrenal androgens and thus have been used to induce ovulation in women with PCOS.110,118 Although a trial of dexamethasone use in 230 clomiphene-resistant women showed efficacy in ovulation and pregnancy rates,

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glucocorticoids are currently not recommended for treatment of infertility.118,141 Surgical intervention with laparoscopic ovarian drilling (LOD) can restore ovulation in 50% of women. In clomiphene-resistant women, LOD is associated with higher pregnancy and ovulation rates that metformin. There is, however, no difference in live birth rates between LOD and either clomiphene citrate combined with metformin or gonadotropin treatment.110,118 In addition, there is risk of developing pelvic adhesions with this procedure. Pregnancy Complications in PCOS

Even after pregnancy is achieved, women with PCOS face greater challenges in achieving a successful outcome from the pregnancy. Rates of early pregnancy loss or miscarriage have been reported as high as 40% in these women compared with 14.3% in normal fertile women.142,143 Possible culprits for this high rate of pregnancy loss include the use of ovulation inducing agents, elevated LH levels, increased androgen levels, insulin resistance, and obesity. Several studies also suggest poor folliculogenesis, resulting in compromised oocyte quality, as a cause for failed pregnancies.144,145 The complex process of follicular and oocyte maturation involves cross-talk between cumulus cells and the oocyte. Gradually this develops the microenvironment that allows the oocyte to gain competence to undergo fertilization and embryogenesis. It is believed that the regulatory system governing follicular development and oocyte maturation may be altered in the setting of PCOS. Pregnancy outcomes in PCOS are also affected by increased risk of late gestational complications. A meta-analysis showed a significantly higher rate of gestational diabetes mellitus (GDM) in women with PCOS. These women, however, also had a higher rate of obesity, which may contribute to the increased risk. Most studies indicate a higher risk of preeclampsia or hypertensive disorders in PCOS pregnancies. Many of these studies, however, are limited by small numbers of subjects. Insulin resistance in the first trimester seems to be associated with an increased risk of preeclampsia. Whether PCOS is associated with preterm birth and low birth weight in infants is unclear. These complications are associated with increased neonatal morbidity and 2.3 times higher rate of admission to the neonatal ICU for PCOS offspring. A metaanalysis indicates a 3-times higher incidence of perinatal mortality due to causes, such as cervical insufficiency, sepsis, and placental abruption.142,143 These complications, however, are also noted with obesity, which often coexists with PCOS. No significant differences have been noted in neonatal malformations from these pregnancies. Because diabetes and obesity often accompany PCOS, it is difficult to dissect the effects of hyperglycemia and adiposity from that of insulin resistance on follicular development and pregnancy outcomes. A recent study, however, in mouse models of varying degrees of insulin resistance in lean, euglycemic mice showed increased antral follicles, abnormal oocyte morphology, increased early pregnancy loss, and low embryonic birth weights in the insulin-resistant mice in comparison with wild type.56 This suggests that insulin resistance or hyperinsulinemia independently affects parameters of female reproduction and gestation. It is, thus, not surprising that the insulin sensitizer, metformin, has been offered as an agent to minimize pregnancy complications. Thus far, studies and meta-analyses have not shown any adverse effects or increased risk of malformations from the use of metformin throughout pregnancy.142 Metformin use during early pregnancy seems to decrease the rate of miscarriages and GDM in women with PCOS.146,147 Use of metformin throughout pregnancy also reduces first-trimester miscarriages and development of diabetes in gestation.148,149


DIABETES IN PREGNANCY

Diabetes in pregnancy remains a significant contributor to maternal and fetal morbidity—affecting 6% to 7% of all pregnancies in the United States.150,151 Pregnancy-related diabetes falls into 2 categories—GDM and pre-GDM. GDM, representing 85% of the cases, manifests in the second to third trimesters of pregnancy. Late gestation is a period of metabolic stress and increased insulin resistance, challenging those with susceptibility to the development of diabetes. This effect is largely attributed to placental secretion of diabetogenic hormones, including growth hormone, corticotropin-releasing hormone, placental lactogen, and corticotropin. Diabetes develops when pancreatic insulin secretion fails to overcome the physiologic insulin resistance.150,151 This subcategory of maternal diabetes is more prevalent in certain ethnic groups, such as Asians, African Americans, Native Americans, and Hispanic individuals compared with the non-Hispanic white population. The category of pre-GDM includes preexisting type 1 diabetes mellitus and type 2 diabetes mellitus. Here, hyperglycemia is seen prior to and throughout the pregnancy. Hyperglycemia typically worsens as pregnancy progresses due to the increasingly insulin-resistant hormonal milieu (described previously). If women do not carry the diagnosis of type 1 diabetes mellitus or type 2 diabetes mellitus, they are screened for diabetes at their first prenatal visit. If the fasting plasma glucose level is greater than 126 mg/dL or HbA1c is greater than 6.5, patients are diagnosed with diabetes and managed accordingly. If the random glucose level exceeds 200 mg/dL, the diagnosis of diabetes is confirmed with an abnormal fasting glucose or HbA1c level. If the initial results are negative, both the American Diabetes Association (ADA) and American Congress of Obstetricians and Gynecologists (ACOG) recommend a 2-step approach to the diagnosis of GDM. A 50-g OGTT at weeks 24 to 28 of gestation is followed by a 100-g, 3-hour OGTT if the 1-hour glucose value is greater than 140 mg/dL during the glucose challenge. Glucose values exceeding the recommended levels at 2 or more time points of the 3-hour test signify a positive result. Two different sets of diagnostic criteria for GDM with the 100-g 3-hour glucose tolerance test have been proposed. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus and the National Diabetes Data Group (NDDG) have proposed threshold values of 105 mg/dL, 190 mg/dL, 165 mg/dL, and 140 mg/dL at baseline and the hourly testing time points, respectively. The Fourth International WorkshopConference on Gestational Diabetes has proposed lower threshold values based on the Carpenter and Coustan modifications: 95 mg/dL, 180 mg/dL, 155 mg/dL, and 140 mg/dL at baseline, 1, 2, and 3 hours after glucose challenge.152 Internationally, however, a 1-step approach is widely practiced using a 75-g OGTT with 2-hour venous sampling. The World Health Organization (WHO) recommends a diagnosis of diabetes if the fasting glucose level is greater than 125 mg/dL or the 2-hour value is greater than 140 mg/dL. The International Association of Diabetes and Pregnancy Study Groups (IADPSG) and the ADA present slightly different cutoff values at fasting, 1-hour, and 2-hour time points (Table 2). Early screening for GDM is recommended in women with strong risk factors, including obesity, family history of diabetes, personal history of GDM, prior stillbirth, and neonate weight greater than 4500 g.150,151 Once a diagnosis of diabetes is ascertained, it is important to counsel those with both GDM and pre-GDM about the increased risk of adverse outcomes for both mother and fetus. Pre-GDM, both type 1 and type 2, can lead to difficulties with fertilization and progression of early pregnancy. Diabetes may affect development of oocyte competence by altering mitchondrial function and altering cell-to-cell interactions between the cumulus cells and the oocyte.153 Periconceptional hyperglycemia

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Table 2 Positive screening criteria for gestational diabetes mellitus 2-h 75-g WHO Fasting

125 mg/dL

1h 2h

140 mg/dL

OGTT IADPSG

3-h 100-g Carpenter/Couston

OGTT NDDG

92 mg/dL

95 mg/dL

105 mg/dL

180 mg/dL

180 mg/dL

190 mg/dL

153 mg/dL

3h

155 mg/dL

165 mg/dL

140 mg/dL

145 mg/dL

can significantly alter organogenesis and lead to malformations in the fetus.154 Congenital malformations have a 4-fold to 10-fold higher incidence in diabetic pregnancies. The most common abnormalities are found in the cardiovascular, central nervous system, gastrointestinal system, urinary tract, caudal regression syndrome, and cleft and palate deformities. Both gestational and pre-GDM conveys an increased risk of developing preeclampsia, miscarriage, and cesarean delivery. Miscarriage occurs at a 3-fold to 5-fold higher rate in diabetic pregnancies than in normal gestations. Fetal outcomes show increased risk of macrosomia, organomegaly, and neonatal respiratory and metabolic complications, such as hypoglycemia, hypocalcemia, and hyperbilirubinemia.155,156 Longer-term effects on the children from diabetic pregnancies may include increased risk of childhood obesity and development of diabetes (Table 3).157–159 The precise mechanism by which hyperglycemia exerts its teratogenic effects is not fully known. Several theories have been proposed. First, glucose is freely transportable

Table 3 Effects of glucose dysregulation on female reproductive function Insulin Resistance Puberty

Premature menarche

Type 1 Diabetes Mellitus

Type 2 Diabetes Mellitus

Delayed menarche

Premature menarche

Gestational Diabetes Mellitus

Ovulation Anovulatory cycles Anovulatory cycles Anovulatory cycles Hyperandrogenism Hyperandrogenism Hyperandrogenism Acanthosis Acanthosis nigricans nigricans Pregnancy Decreased fertility

Decreased fertility

Decreased fertility

Maternal

Preeclampsia Miscarriage Cesarean delivery GDM

Preeclampsia Miscarriage Cesarean delivery



Fetal

Preterm birth Macrosomia Low birth weight Organomegaly Neonatal mortality Respiratory complications Hypoglycemia Hyperbilirubinemia Shoulder dystocia Congenital anomalies Malformations

Macrosomia Organomegaly Respiratory complications Hypoglycemia Hyperbilirubinemia Shoulder dystocia Congenital anomalies Malformations

Macrosomia Organomegaly Respiratory complications Hypoglycemia Hyperbilirubinemia Shoulder dystocia


across the placenta. Hyperglycemia or the resultant hyperinsulinemia in the embryo may mediate some of these effects. For example, the heart has high levels of insulin receptors. Hypertrophic myopathy can be seen in newborns from diabetic mothers that, at times, regresses after birth. Excess insulin can also delay pulmonary maturation by decreased surfactant production. A second theory suggests that generation of reactive oxygen species leads to damage of the developing yolk sac. Epigenetic changes in diabetic pregnancies may also affect organogenesis and fetus viability. Because of the high rate of complications in the setting of hyperglycemia, the ADA currently recommends stricter glycemic control, with an HbA1c goal of less than 6.0%, prior to conception and throughout pregnancy. The importance of glucose control in improving pregnancy outcomes was demonstrated in 2 large prospective randomized controlled trials.151 The Australian Carbohydrate Intolerance Study in Pregnant Women is a multicenter, 10-year study conducted in 14 sites in Australia, evaluating the benefits of glucose control in mild GDM in 1000 women.160 Treatment was associated with a significant reduction in the primary outcome of perinatal complications (including perinatal death, shoulder dystocia, and birth trauma), with an adjusted relative risk of 0.33% and 95% CI of 0.14–0.75. In secondary outcomes, treatment did reduce the rate of large for gestational age neonates and maternal preeclampsia but failed to show differences in rates of neonatal hypoglycemia, jaundice, or respiratory difficulties. A second study evaluating treatment of mild GDM in 958 women was conducted by the National Institute of Child Health and Human Development Maternal-Fetal Medicine Units. The primary outcomes of perinatal death, neonatal hypoglycemia, and elevated cord C-peptide did not show any significant differences between the 2 groups. There was an improvement in secondary outcomes, however, including lower frequency of fetal overgrowth, development of neonatal fat, risk of shoulder dystocia, cesarean delivery, and preeclampsia.161 The initial approach to treatment of diabetes in pregnancy consists of lifestyle counseling and dietary changes. The recommendations include a diet of 2000 kcal to 2500 kcal daily intake, with carbohydrates comprising 33% to 40% of calories.162,163 Three meals and 2 to 3 snacks allow distribution of glucose intake without significant elevations in postprandial glucose excursions. The Institute of Medicine recommends a weight gain of 5 kg to 19 kg dependent on the maternal pregestational BMI.164 Although regular exercise improves insulin resistance and is recommended as an adjunct to dietary changes, studies evaluating exercise in GDM have shown mixed results.165 Daily self–glucose monitoring is an important component of diabetes management in pregnancy. The ADA recommends target glucose values of fasting less than 95 mg/dL, 1-hour postprandial less than 140 mg/dL, and 2-hour postprandial less than 120 mg/dL. The ACOG recommends fasting glucose less than 95 mg/dL, 1-hour postprandial less than 130 mg/dL to 140 mg/dL, and 2-hour postprandial less than 120 mg/dL. Approximately 25% of women with GDM require medication. The mainstay of treatment has been the use of neutral protamine Hagedorn (NPH) and regular insulin to provide basal and bolus dosing. The newer classes of insulin analogs, although widespread in practice in the general population, must be used with caution in the pregnant diabetic woman. The insulin analogs have alterations in the amino acid sequence or post-translational modification that lead to decreased or increased affinity for the IGF-1 receptor. IGF-1 is a critical player in embryo maturation, implantation, and pregnancy progression. Therefore, well-controlled, randomized, and long-term studies are needed to assure the safety of using insulin analogs in pregnancy.166 The first study to indicate the safety and efficacy of insulin lispro was published in 1999.167 Lispro was successful in lowering postprandial glucose excursions and

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HbA1c levels similar to regular insulin while producing fewer episodes of hypoglycemia. Similarly, in a large clinical trial with 213 patients randomized to receive either lispro or regular insulin, no significant differences were noted in maternal or fetal outcomes.168,169 There is some concern that although insulin does not seem to cross the placental barrier, insulin bound to immunoglobulin can do so and potentially lead to diabetic fetopathy. In both of these studies, however, lispro was not associated with a greater increase in anti-insulin antibody levels. Short-acting insulin aspart was introduced in 1999. Initial studies suggest the safety and efficacy of this insulin analog for use in women with GDM.170–172 Higher patient satisfaction was noted with the shortacting insulin analogs in comparison with regular insulin use. Insulin detemir and insulin glargine are long-acting insulin analogs, the latter with a 6-fold increase in affinity for IGF-1 receptor in comparison with human insulin.166 There have not yet been extensive randomized controlled trials in pregnant women with either of these agents. A meta-analysis of observational studies, including 331 pregnancies with use of glargine during the first, second, or third trimester, did not show any increase in adverse fetal and maternal outcomes with glargine versus NPH insulin. The number of women in these studies using glargine in the first trimester was, however, too small to assess an effect on congenital malformations.173 In 2012, insulin detemir was revised as a category B pregnancy therapeutic from pregnancy category C. This reclassification was based on a large ramdomized trial in women with type 1 diabetes mellitus given either NPH or insulin detemir treatment. Insulin detemir and NPH achieved similar HbA1c and hypoglycemia rates. No increase in adverse maternal or fetal health events has been noted.174 One limiting factor, however, with basal insulin is the steady rate of insulin release. It may be more difficult to mimic the diurnal variations in glucose range, with increased insulin resistance and hyperglycemia occurring in the morning hours. Insulin pump device as a modality for insulin delivery is increasingly used to treat pregnant women. Theoretically, this approach may allow more rapid adjustments in basal as well as bolus insulin regimen as insulin needs change throughout the pregnancy. A review of 6 randomized trials comparing the use of the traditional multiple daily injections versus continuous subcutaneous insulin infusion, however, found no significant differences between the 2 groups in terms of glucose control or maternal and fetal outcomes.175 It is important to be aware of the variations in insulin requirement throughout gestation. Observational studies suggest an early rise in insulin needs in weeks 3 to 7, followed by a decrease in insulin requirements in the second trimester. The increased production of placental hormones as the pregnancy progresses, however, increases gestational hyperglycemia. The average insulin requirement in type 1 diabetes mellitus progresses from 0.7 U/kg in the first trimester to 0.8 U/kg in the second trimester, 0.9 U/kg in weeks 29 to 34, and 1.0 U/kg after week 35. These ratios should be individualized. Obese insulin-resistant women may require up to 1.5 U/kg to 2.0 U/kg in initial dosing.176 At times, glucose values can decrease after week 35, especially in women with type 1 diabetes mellitus. A fall in insulin dosing of 5% to 10% should instigate a review of fetal health. Up to 30% reduction in insulin requirements, however, can also be seen in healthy pregnancies.177 Oral hypoglycemic agents, such as glyburide and metformin, have traditionally been excluded from the treatment armamentarium for diabetes in pregnancy because of concern regarding fetal teratogenicity and neonatal hypoglycemia. These concerns stem from the lack of certainty about crossing the placental barrier. In a recent meta-analysis reviewing randomized controlled trials comparing oral hypoglycemic agents with insulin use in diabetes of pregnancy, few differences were noted in both


fetal and maternal outcomes. There were no significant differences in glucose control between the 2 groups, either in fasting glucose or postprandial glucose excursion. There was no increase with use of an oral hypoglycemic agent in neonatal hypoglycemia, birthweight, admission to neonatal ICU, respiratory distress, preterm births, congenital anomalies, or intrauterine fetal death. With maternal complications, there was no significant difference in the rate of cesarean sections, but there was a lower rate of hypoglycemia and gestational hypertension in the oral hypoglycemic cohorts.178 A second systematic review by Nicholson and colleagues179 of randomized controlled trials and observational studies confirmed these results. Differences in study design and lack of long-term outcome data make it difficult, however, to endorse the use of oral hypoglycemic agents in the treatment of diabetes in pregnancy. Glyburide, a second-generation sulfonylurea, is the oral antidiabetic agent most commonly used in diabetic pregnancies. Langer and colleagues180 conducted a large randomized trial with 404 women who received insulin or glyburide. Similar improvements were noted in hyperglycemia, whereas there were no differences seen in macrosomia, neonatal hypoglycemia, neonatal morbidities, or cord insulin concentrations. Of note, 4% of the women treated with glyburide ultimately needed insulin in the treatment of maternal diabetes. A smaller study, however, demonstrated higher mean glucose levels in women treated with glyburide versus insulin.181 Metformin, a biguanide, has also been used in the treatment of diabetes in pregnancy. Although metformin is able to cross the placenta, it does not seem to have teratogenic effects. The largest study assessing the use of metformin in 751 Australian women given biguanide or insulin at weeks 20 to 33 of gestation reported similar perinatal morbidity in the 2 groups. No serious adverse outcomes were found in the metformin group. However, 46% of women receiving metformin required supplemental insulin to achieve glycemic control.181 A randomized controlled trial comparing metformin with glyburide use in GDM suggests that glyburide is more effective in achieving adequate glycemic control—16% of the glyburide group and 35% of the metformin group required supplemental insulin.182 The ADA and the ACOG, however, do not endorse the use of either of these agents in the treatment of diabetes in pregnancy. There are currently no controlled trials using other hypoglycemic agents, such as thiazolidinediones, a-glucosidase inhibitors, glitinides, or GLP-1 agonists in pregnancy. Women with GDM have a high risk of recurrence in subsequent pregnancies, as high as 41.3%.180 There seems to be an increased risk in women with increased age, multiparous state, greater prepregnancy weight, and higher infant birth weight.183 One third of women with GDM continue to have overt diabetes, impaired fasting glucose, or impaired glucose tolerance during the postpartum period. Both the ADA and ACOG recommend screening with fasting plasma glucose and 2-hour, 75-g OGTT at 6 to 12 weeks postpartum.180 If this screening is found negative, assessment of glycemic status is recommended every 3 years. The relative risk of developing type 2 diabetes mellitus is 4.69 within the first 5 years after gestation and 9.34 more than 5 years after delivery.184 The presence of islet cell antibodies, specific HLA alleles (DR3 or DR4), and development of diabetic ketoacidosis predisposes the mother to development of type 1 diabetes mellitus postpartum.185,186 Careful postpartum monitoring in GDM is important in decreasing maternal morbidity. SUMMARY

In summary, the insulin/IGF pathways and glucose metabolism act as key mediators of human ovarian function and female fertility. In the setting of normal insulin action, insulin binds to its own receptors in the ovary to mediate steroidogenesis and act

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as a co-gonadotropin. Insulin in synergy with other factors, such as hCG, may also influence ovarian growth and cyst formation. The IGF pathway also seems to influence normal ovarian function. Insulin signaling affects reproductive function through brainspecific or central action as well as direct gonadal action. The importance of insulin/IGF action in female reproductive function is conserved throughout various species. Dysregulation of this pathway leads to altered puberty, ovulation, and fertility. When fertility is achieved, there remain risks to both maternal health and fetal development (see Table 3). Better understanding of the normal physiology and pathophysiology of insulin, IGF, and glucose effects on the human reproductive system will allow for better outcomes in affected women. REFERENCES

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Marijuana, the Endocannabinoid System and the Female Reproductive System.

The Krüppel-like factors in female reproductive system pathologies.

Telocytes in female reproductive system (human and animal).

Diabetes-induced alterations of reproductive and adrenal function in the female rat.

Function and regulation of MTA1 and MTA3 in malignancies of the female reproductive system.

Nonproliferative and proliferative lesions of the rat and mouse female reproductive system.

Adipokines and the female reproductive tract.

Metals and female reproductive toxicity.

Scientific principles of regenerative medicine and their application in the female reproductive system.

Ultra- and microstructure of the female reproductive system of Matsucoccus matsumurae.

The Effects of Chlorpromazine on Reproductive System and Function in Female Rats.

Growth hormone production and role in the reproductive system of female chicken.

Inhibition of the reproductive system by deslorelin in male and female pigeons (Columba livia).

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system.

Effect of spinal cord transection on the reproductive system in the female rat.

Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations.

[Comparative histological study of the reproductive system of the female llama (Lama guanicoe glama). I. Ovary].

Neonatally induced mild diabetes: influence on development, behavior and reproductive function of female Wistar rats.

carbon monoxide in the female reproductive system: an overlooked signalling pathway.

Association of Female Reproductive Factors with Hypertension, Diabetes and LQTc in Chinese Women.

Cell-specific regulation of progesterone receptor in the female reproductive system.

Steroid receptor coactivators as therapeutic targets in the female reproductive system.

Melatonin treatment delays reproductive aging of female rat via the opiatergic system.

Effect of 1,25-dihydroxyvitamin D3 on the female reproductive system in rats.